Molecular surveillance of invasive penicillin-resistant Streptococcus pneumoniae Colombian isolates recovered from children less than 5 years of age

The rapid increase of penicillin-resistant Streptococcus pneumoniae isolates could be a consequence of the spread of clones or due to the antimicrobial selective pressure. The genetic relatedness of 190 invasive isolates S. pneumoniae with reduced susceptibility to penicillin recovered from Colombian children less than 5 years old during a surveillance study from 2000 to 2003 was determined by the use of pulsed-field electrophoresis (PFGE). Overall, 42 different PFGE patterns were identified, but 4 of them included 76% of all isolates. They were related with international clones 1-Spain23F, 2-Spain6B, 3-Spain9V and 26-Colombia23F. Our results indicated that the dissemination of penicillin-resistant S. pneumoniae was the result of the spread of international clones, specially, the 3-Spain9V clone.     

Population structure of invasive Streptococcus pneumoniae in The Netherlands in the pre-vaccination era assessed by MLVA and capsular sequence typing

The introduction of nationwide pneumococcal vaccination may lead to serotype replacement and the emergence of new variants that have expanded their genetic repertoire through recombination. To monitor alterations in the pneumococcal population structure, we have developed and utilized Capsular Sequence Typing (CST) in addition to Multiple-Locus Variable number tandem repeat Analysis (MLVA).To assess the serotype of each isolate CST was used. Based on the determination of the partial sequence of the capsular wzh gene, this method assigns a capsular type of an isolate within a single PCR reaction using multiple primersets. The genetic background of pneumococcal isolates was assessed by MLVA. MLVA and CST were used to create a snapshot of the Dutch pneumococcal population causing invasive disease before the introduction of the 7-valent pneumococcal conjugate vaccine in The Netherlands in 2006. A total of 1154 clinical isolates collected and serotyped by the Netherlands Reference Laboratory for Bacterial Meningitis were included in the snapshot. The CST was successful in discriminating most serotypes present in our collection. MLVA demonstrated that isolates belonging to some serotypes had a relatively high genetic diversity whilst other serotypes had a very homogeneous genetic background. MLVA and CST appear to be valuable tools to determine the population structure of pneumococcal isolates and are useful in monitoring the effects of pneumococcal vaccination.     

Serotype distribution of invasive Streptococcus pneumoniae in Canada during the introduction of the 13-valent pneumococcal conjugate vaccine, 2010

A baseline serotype distribution was established by age and region for 2058 invasive Streptococcus pneumoniae isolates collected during the implementation period of the 13-valent pneumococcal conjugate vaccine (PCV13) program in many parts of Canada in 2010. Serotypes 19A, 7F, and 3 were the most prevalent in all age groups, accounting for 57% in <2 year olds, 62% in 2-4 year olds, 45% in 5-14?year olds, 44% in 15-49?year olds, 41% in 50-64?year olds, and 36% in ≥65?year olds. Serotype 19A was most predominant in Western and Central Canada representing 15% and 22%, respectively, of the isolates from those regions, whereas 7F was most common in Eastern Canada with 20% of the isolates. Other prevalent serotypes include 15A, 23B, 12F, 22F, and 6C. PCV13 serotypes represented 65% of the pneumococci isolated from?<2 year olds, 71% of 2-4 year olds, 61% of 5-14 year olds, 60% of 15-49 year olds, 53% of 50-64 year olds, and 49% of the?≥65?year olds. Continued monitoring of invasive pneumococcal serotypes in Canada is important to identify epidemiological trends and assess the impact of the newly introduced PCV13 vaccine on public health.     

侵袭性和非侵袭性肺炎链球菌耐药率的比较分析

目的研究侵袭性和非侵袭性肺炎链球菌的耐药谱的差异,指导合理应用抗生素及感染管理。方法回顾性统计分析2009至2011年来天台县人民医院就诊患者分离肺炎链球菌的标本来源及耐药性,比较侵袭性和非侵袭性肺炎链球菌耐药率之间的差异。结果共分离出肺炎链球菌642株,痰液中分离出584株,非痰液中分离出58株,其中血液中分离出32株,脑脊液中分离出20株,其他分离出6株,所有肺炎链球菌对万古霉素和利奈唑胺均敏感,对青霉素、红霉素、克林霉素、四环素及复方新诺明耐药严重,对左氧氟沙星、氯霉素比较敏感;侵袭性分离株对青霉素、红霉素、克林霉素、左氧氟沙星、四环素及氯霉素的耐药率显著高于非侵袭性肺炎链球菌。结论该院分离的肺炎链球菌主要来自痰液标本,耐青霉素肺炎链球菌的检出率高,大环内酯类耐药严重,存在一定比例的侵袭性感染,非侵袭菌株与侵袭性菌株耐药谱之间存在一定差异,临床治疗应该区别对待,系统的监测细菌耐药性,合理选择抗菌药物。     

Extracellular matrix formation enhances the ability of Streptococcus pneumoniae to cause invasive disease

During infection, pneumococci exist mainly in sessile biofilms rather than in planktonic form, except during sepsis. However, relatively little is known about how biofilms contribute to pneumococcal pathogenesis. Here, we carried out a biofilm assay on opaque and transparent variants of a clinical serotype 19F strain WCH159. After 4 days incubation, scanning electron microscopy revealed that opaque biofilm bacteria produced an extracellular matrix, whereas the transparent variant did not. The opaque biofilm-derived bacteria translocated from the nasopharynx to the lungs and brain of mice, and showed 100-fold greater in vitro adherence to A549 cells than transparent bacteria. Microarray analysis of planktonic and sessile bacteria from transparent and opaque variants showed differential gene expression in two operons: the lic operon, which is involved in choline uptake, and in the two-component system, ciaRH. Mutants of these genes did not form an extracellular matrix, could not translocate from the nasopharynx to the lungs or the brain, and adhered poorly to A549 cells. We conclude that only the opaque phenotype is able to form extracellular matrix, and that the lic operon and ciaRH contribute to this process. We propose that during infection, extracellular matrix formation enhances the ability of pneumococci to cause invasive disease.     

人工智能时代发酵优化与放大技术的机遇与挑战

人工智能(artificial intelligence, AI)技术正引发一场新的产业革命,其成功应用正从信息产业迅速渗透到各行各业。传统的发酵工程技术受到巨大挑战的同时更多地迎来了发展变革的机遇。首先,合成生物技术飞速发展使高性能菌株的可获得性及获取效率显著提升,对传统低效的发酵优化放大技术提出很大挑战,亟需对发酵优化放大技术进行升级,以满足高通量菌种性能验证及工艺开发能力的需求;其次,发酵装备技术的持续发展为高效发酵优化技术的进步奠定了良好基础,加之人工智能技术特别是数字孪生与知识图谱等技术的应用,将为传统发酵技术的颠覆性发展带来巨大推动力。本文分别从合成生物时代对发酵优化技术的挑战、发酵优化与放大的核心技术、高通量发酵装备技术、数据可视化技术、数字孪生及知识图谱等智能技术在发酵优化放大中的应用等几个方面进行综述,并对未来工业发酵优化技术的场景以及未来发酵技术对人才培养等提出的新要求进行了展望。     

Evidence of conformational switch in Streptococcus pneumoniae FtsZ during polymerization

FtsZ, the master coordinator of bacterial cell division, assembles into filaments in the presence of nucleotide. FtsZ from Streptococcus pneumoniae bears two tryptophan residues (W294 and W378) in its amino acid sequence. The tryptophan fluorescence of FtsZ increases during the assembly of FtsZ. We hypothesized that this increase in the fluorescence intensity was due to the change in the environment of one or both tryptophan residues. To examine this, we constructed two mutants (W294F and W378F) of FtsZ by individually replacing tryptophan with phenylalanine. The mutants displayed similar secondary structures, GTPase activity, and polymerization ability as the wild type FtsZ. During the polymerization, only one tryptophan (W294) showed an increase in its fluorescence intensity. Using time‐correlated single‐photon counting, the fluorescence lifetime of W294 was found to be significantly higher than W378, indicating that W294 was more buried in the structure than W378. The lifetime of W294 further increased during polymer formation, while that of W378 remained unchanged. Fluorescence quenching experiment suggested that the solvent exposure of W294 reduced during the polymerization of FtsZ. W294 is located near the T‐7 loop of the protein, a region important for the monomer‐monomer interaction during the formation of a protofilament. The results indicated that the region around W294 of S. pneumoniae FtsZ undergoes a conformational switch during polymerization as seen for FtsZ from other bacteria.     

Versatility of the capsular genes during biofilm formation by Streptococcus pneumoniae

Streptococcus pneumoniae forms part of the natural microbiota of the nasopharynx. For the pneumococcus to cause infection, colonization needs to occur and this process is mediated by adherence of bacteria to the respiratory epithelium. Although the capsular polysaccharide (CPS) of S. pneumoniae is known to be important for infection to occur, its role in colonization is controversial. Biofilm models are starting to emerge as a promising tool to investigate the role of CPS during nasopharyngeal carriage, which is the first step in the dissemination and initiation of a pneumococcal infection. Using a well-defined model system to analyse in vitro biofilm formation in pneumococcus, here we explore the molecular changes underlying the appearance of capsular mutants using type 3 S. pneumoniae cells. Spontaneous colony phase variants show promoter mutations, as well as duplications, deletions and point mutations in the cap3A gene, which codes for a UDP-glucose dehydrogenase (UDP-GlcDH). Increased biofilm-forming capacity could usually be correlated with a reduction both in colony size and in the relative amount of CPS present on the cell surface of each colony variant. However, a mutation in Cap3A Thr83Ile (a strictly conserved residue in bacterial UDP-GlcDHs) that resulted in very low CPS production also led to impaired biofilm formation. We propose that non-encapsulated mutants of pneumococcal type 3 strains are essentially involved in the initial stages (the attachment stage) of biofilm formation during colonization/pathogenesis.     

Commonly invasive serotypes of Streptococcus pneumoniae trigger a reduced innate immune response compared with serotypes rarely responsible for invasive infection

Although there are more than 90 serotypes of Streptococcus pneumoniae (or pneumococcus), it is not understood why a small number of serotypes account for most invasive infections. To investigate the human innate immune response triggered by different pneumococcal serotypes, monocyte-derived macrophages were exposed to a group of commonly and rarely invasive pneumococcal clinical isolates and tumor necrosis factor (TNF)-alpha production was measured. Commonly invasive pneumococcal serotypes triggered significantly less TNF-alpha production than serotypes rarely responsible for invasive infection (P<0.004). These data indicate that one factor influencing the invasive potential of a pneumococcal serotype is the magnitude of innate immune-mediated TNF-alpha production triggered by exposure to the organism and suggest that the integrated host response generated against commonly invasive pneumococcal serotypes may be less effective than the response directed against rarely invasive serotypes.     

Utilization of putrescine by Streptococcus pneumoniae during growth in choline-limited medium

Polyamines such as putrescine are small, ubiquitous polycationic molecules that are required for optimal growth of eukaryotic and prokaryotic cells. These molecules have diverse effects on cell physiology and their intracellular content is regulated by de novo synthesis and uptake from the environment. The studies presented here examined the structure of a putative polyamine transporter (Pot) operon in Streptococcus pneumoniae (pneumococcus) and growth of pneumococci in medium containing putrescine substituted for choline. RT-PCR experiments demonstrated that the four genes encoding the Pot system are co-transcribed with murB, a gene involved in an intermediary step of peptidoglycan synthesis. Pneumococci grown in chemically-defined media (CDM) containing putrescine without choline enter logarithmic phase growth after 36-48 hs. However, culture density at stationary phase eventually reaches that of choline-containing medium. Cells grown in CDM-putrescine formed abnormally elongated chains in which the daughter cells failed to separate and the choline-binding protein PspA was no longer cell-associated. Experiments with CDM containing radiolabeled putrescine demonstrated that pneumococci concentrate this polyamine in cell walls. These data suggest that pneumococci can replicate without choline if putrescine is available and this polyamine may substitute for aminoalcohols in the cell wall teichoic acids.     

Fate of DNA in eclipse complex during genetic transformation in Streptococcus pneumoniae

Uptake of DNA and genetic recombination proceeded normally in competent Streptococcus pneumoniae despite inhibition of DNA replication by 6-(p-hydroxyphenylazo)-uracil. Immediately after a brief uptake period, 68% of donor DNA label was in eclipse complex form, and 22% was in low-molecular-weight products; by the completion of integration at 10 min, 23% was integrated into the chromosome, and the rest was lost from the cell. Throughout the process, less than 1% was found as free single strands. The DNA in eclipse complex is therefore an intermediate in the integration process.     

Nasopharyngeal colonization and invasive disease are enhanced by the cell wall hydrolases LytB and LytC of Streptococcus pneumoniae

Background

Streptococcus pneumoniae is a common colonizer of the human nasopharynx and one of the major pathogens causing invasive disease worldwide. Dissection of the molecular pathways responsible for colonization, invasion, and evasion of the immune system will provide new targets for antimicrobial or vaccine therapies for this common pathogen.

Methodology/Principal Findings

We have constructed mutants lacking the pneumococcal cell wall hydrolases (CWHs) LytB and LytC to investigate the role of these proteins in different phases of the pneumococcal pathogenesis. Our results show that LytB and LytC are involved in the attachment of S. pneumoniae to human nasopharyngeal cells both in vitro and in vivo. The interaction of both proteins with phagocytic cells demonstrated that LytB and LytC act in concert avoiding pneumococcal phagocytosis mediated by neutrophils and alveolar macrophages. Furthermore, C3b deposition was increased on the lytC mutant confirming that LytC is involved in complement evasion. As a result, the lytC mutant showed a reduced ability to successfully cause pneumococcal pneumonia and sepsis. Bacterial mutants lacking both LytB and LytC showed a dramatically impaired attachment to nasopharyngeal cells as well as a marked degree of attenuation in a mouse model of colonization. In addition, C3b deposition and phagocytosis was more efficient for the double lytB lytC mutant and its virulence was greatly impaired in both systemic and pulmonary models of infection.

Conclusions/Significance

This study confirms that the CWHs LytB and LytC of S. pneumoniae are essential virulence factors involved in the colonization of the nasopharynx and in the progress of invasive disease by avoiding host immunity.     

Persistent high burden of invasive pneumococcal disease in South African HIV-infected adults in the era of an antiretroviral treatment program

Background

Highly active antiretroviral treatment (HAART) programs have been associated with declines in the burden of invasive pneumococcal disease (IPD) in industrialized countries. The aim of this study was to evaluate trends in IPD hospitalizations in HIV-infected adults in Soweto, South Africa, associated with up-scaling of the HAART program from 2003 to 2008.

Methods

Laboratory-confirmed IPD cases were identified from 2003 through 2008 through an existing surveillance program. The period 2003-04 was designated as the early-HAART era, 2005–06 as the intermediate-HAART era and 2007–08 as the established-HAART era. The incidence of IPD was compared between the early-HAART and established-HAART eras in HIV-infected and–uninfected individuals.

Results

A total of 2,567 IPD cases among individuals older than 18 years were reported from 2003 through 2008. Overall incidence of IPD (per 100,000) did not change during the study period in HIV-infected adults (207.4 cases in the early-HAART and 214.0 cases in the established-HAART era; p = 0.55). IPD incidence, actually increased 1.16-fold (95% CI: 1.01; 1.62) in HIV-infected females between the early-and established-HAART eras (212.1 cases and 246.2 cases, respectively; p = 0.03). The incidence of IPD remained unchanged in HIV-uninfected adults across the three time periods.

Conclusion

Despite a stable prevalence of HIV and the increased roll-out of HAART for treatment of AIDS patients in our setting, the burden of IPD has not decreased among HIV-infected adults. The study indicates a need for ongoing monitoring of disease and HAART program effectiveness to reduce opportunistic infections in African adults with HIV/AIDS, as well as the need to consider alternate strategies including pneumococcal conjugate vaccine immunization for the prevention of IPD in HIV-infected adults.     

Resistance mechanism of chloramphenicol in Streptococcus haemolyticus, Streptococcus pneumoniae and Streptococcus faecalis.

The chloramphenicol resistance of Streptococcus haemolyticus, Streptococcus pneumoniae and Streptococcus faecalis isolated from clinical materials was proved to be due to an inactivating enzyme produced by these bacteria. The inactivated products of chloramphenicol were identified as 1-acetoxy, 3-acetoxy and 1,3-diacetoxy derivatives by thin-layer chromatography and infrared spectroscopy. The responsible enzyme was thus confirmed to be chloramphenicol acetyltransferase. The enzyme was inducible. It was partially purified by ammonium sulfate precipitation, DEAE-cellulose chromatography and gel filtration on Sephadex G-150. The enzymes obtained from S. haemolyticus, S. pneumoniae and S. faecalis have been compared with the conclusion that they are identical with respect to molecular weight (approximately 75,000-80,000), optimum pH and heat stability.     

Modulation of competence for genetic transformation in Streptococcus pneumoniae

The spontaneous development of competence by cultures of Streptococcus pneumoniae in casein hydrolysate medium was strongly dependent on the initial pH of the culture medium. Cells growing in cultures beginning with a wide range of initial pH values (6.8 to 8.0) all developed competence, as measured by [3H]DNA uptake, [3H]DNA degradation and genetic transformation; but the initial pH of the medium affected both the timing of the occurrence of competence and the number of times the culture became competent. In cultures grown in media of lower initial pH, competence occurred only once, at high population densities, while in more alkaline media a succession of competence cycles occurred, beginning at lower cell densities. The critical population density required for the initiation of competence varied tenfold over the pH range studied. Successive competence cycles in an alkaline medium were not equivalent: while the percentage of competent cells in the first competence cycle was high (approximately 80%), that in the second competence cycle was lower (approximately 12%). Correspondingly, competence-specific proteins were less prominent in the labelled-protein pattern of the second competence cycle than in that of the first. These features of the physiology of competence control make it possible to adjust the expression of competence to suit various experimental requirements.